Materials and Methods 2221 Essential oils

Plant Materials: The adult leaves of the eleven Eucalyptus species (E. punctata DC, E. sideroxylon Cunn., E. saligna Smith, E. cladocalyx F. Muell., E. albens Miq.ex Benth., E. ovata Labill., E. leucoxilon F. Muell., E. Blakelyi A. Cunn, E. microcorys F. Muell. camaldulensis DC., and E. globulus Labill) were collected from the trees located in Bainem forest (16 km to the west of Algiers). Two or three trees of each species were taken randomly. Voucher specimens were cut off and housed in the arboretum of the Institute National de la Recherche Forestière (INRF). The oils were isolated from the fresh leaves by steam distillation.

• Oil isolation: The oils were isolated from the fresh leaves by steam distillation for 2.5 hours using a modified clevenger-type apparatus and stored at low temperature. The oil yield of each species was calculated. The following analyses were conducted on the oils: specific gravity (20°C), refractive index (20°C), optical rotation (20°C), ester value and acid value, GC, GC-MS, and GC-FTIR.

• Physicochemical indices: The physicochemical indices of the oils were determined following the ISO regulation. ISO 279:1981 for the specific gravity, ISO 280:1976 for the refractive index, ISO 592:1981 for the optical rotation, ISO 709: 1980 for the ester value and ISO 1242: 1973 for the acid value.

• GC analysis: Each oil was analyzed by GC on a Carlo Erba Gas Chromatograph 5160 using the following experimental conditions: fused silica SE52 column, 30 m x 0.32 mm; column temperature 45°C (6 min) to 250°C at 3°C/min; injection mode split; detector FID; injector and detector temperature, 280°C; carrier gas He 100 KPa; injected volume 1 ^l of solution 1/100 in pentane of the oil.

• GC-MS analysis: Samples were analyzed by GC/MS (EI) on a Fison MD 800 system equipped with Adams' library (15). Two different columns were used: DB5 (30 m x 0.25 mm) and Carbowax 20 M (30 m x 0.32 mm) fused silica columns. The GC condition for the two columns: 60°C (6 min) to 240°C at 3°C/min; injector temperature 250°C; injection mode split, split ratio 1/30; volume injected 1 ^l of solution 1/100 in pentane of the oil; carrier gas He 90 KPa; interface temperature 250°C; detector 1.5 kV; and acquisition mass 41-300 amu.

• GC-FTIR analysis: Samples were analyzed on Nicolet 20SXC system equipped by a MCT detector. The interface GCIR was a light pipe of 100 |l and 1 mm diameter. The chromatograph was a Perkin-Elmer 8500 equipped with a FID detector and a BP1 fused silica column (50 m x 0.32 mm). GC conditions: 60-250°C at 2°C/min.

The identification of components was established by combining the comparison of mass spectra of components with the published spectra (Adams, 1995) and the retention indices with the published index data (Jennings et al., 1980). The quantitative composition was obtained by peak area normalization, the response factor for each component was supposed to be equal to one. For the GC-FTIR, the identification of components was achieved by combining the comparison of spectrum of components with Aldrich-vapor phase (Library spectrum) and a spectrum of condensed compound in a literature especially for the sesquiterpenes. The quantitative composition was obtained by peak area normalization, the response factor for each component was supposed to be equal to one.

22.2.2 Atmospheric emission

Biogenic VOCs emitted from E. globulus were collected using static branch cuvettes. They were made by Tedlar bags, 3 L in volume, equipped with inlet and outlet lines for air circulation. Samples were collected 5 min after branch enclosure to prevent heat stress to the plant leaves. All experiments were carried out under light saturation conditions (PAR > 1000 p.mol m-2 s-1) with leaf temperatures ranging from 27 to 30°C.

Air emission samples from the tree were enriched on glass tubes (15 cm x 0.3 cm I.D.) filled with a bed of Carbopack C (0.034 g) and Carbograph 1 (0.17 g), set in series. Flow rates ranging from 200 to 330 ml were used for sample collection. After sample collection, traps were wrapped in aluminum foil and stored at room temperature until they were subjected to chemical determinations. After removing oxygen and the excess of water from the adsorbents by a back-flushing step, traps were thermally desorbed at 250°C and VOCs cryofocused in an empty liner kept at -120°C. Injection into the capillary column was achieved by fast heating the liner from -150 to +150°C in 10 s.

The separation of desorbed VOCs was performed on a Cyclodex-B capillary column, 30 m x 0.256 mm I.D., 0.25 p.m film thickness supplied by J & W Scientific (CA, USA). Mass spectrometric determinations were performed using a HP 5890 gas chromatograph coupled with a HP 5970B mass-selective detector (Hewlett Packard Instruments, Palo Alto, CA, USA). Thermal desorption of sampled tubes, cryofocusing of released vapors, and their injection into the capillary column were performed with a TCT/PT1 CP4001 unit supplied by Chrompack (Middleburg, The Netherlands).

For peak identification the mass spectrometer was operated in scan conditions by collecting all ions from 20 to 250 m/z. A scan frequency of 3 scans s-1 was used for generating the mass chromatogram. Positive identification was obtained by combining the mass spectral information with the elution sequence obtained through the analysis of pure compounds.

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